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For routine plasmid transformations, where high efficiency is not required, the following method which is a modification of that described by Sambrook and Russell (2001) was used. An overnight culture of the recipient strain was subcultured in fresh LB and grown till mid-exponential phase. The culture was chilled on ice for 15 min, and the steps hereafter were done on ice or at 4°C. The culture was centrifuged, and the pellet was resuspended in one third volume of cold 0.1 M CaCl2. After 15 min incubation on ice, the cells were again recovered by centrifugation, and resuspended in one tenth volume of cold 0.1 M CaCl2. The suspension (0.1 ml) was incubated on ice for 1 h after which DNA was added (~10-100 ng of DNA in less than 10 μl volume). The mixture was again incubated on ice for 30 min, and then heat shocked for 90 seconds at 42°C. Immediately 0.9 ml of LB broth was added to the tube and incubated at 37°C for 45 min for phenotypic expression of the antibiotic marker before being plated on selective medium at various dilutions. A negative control tube (with no plasmid DNA addition) was also routinely included in each of the experiments

For confocal microscopyanalysis, 5X105THP-1 cells were seeded and treatedwithPMA in 4-chambered slides. Differentiated THP-1 macrophageswere infected either with FITC-labeled or GFP-expressingC.glabratastrains to a MOIof 1:1. At different time intervals, medium was aspirated out from each chamber of 4-chambered slides and chamberes were washed twice with PBS. To fixthe infected macrophages,500 μlformaldehyde(3.7%) was added gently toeach chamber andincubated for 15 minat room temperature. Each chamber of the slide was washed twice withPBS to remove formaldehyde solution completely. To permeabilize the fixed cells, 500 μl Triton-X (0.7%) was dispensed toeach chamber and slide wasincubated at room temperature for 5 min. Chambers of the slide werewashed twice with PBS, separated from the slideusing a chamber removal device andwere air dried. Coverslips were placed onslides using Vectashield mounting mediumand bordersweresealed withnail paint. Slides werestored at 4°C until used forfluorescence imaging

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and finally resuspended in 100 μl sterile water. Bacterial cell suspension was aliquoted in 20 μl volume. The above procedure was followed for all the three strains and cell suspension of three different strains were mixed together in 1:1:1 ratio. For conjugation to occur, 20 μl of the above mixture was spottedon the LB agar plate and incubated at 37°C for 12-16 h. Next, the conjugation drops were streaked on LB agar plate containing appropriate antibiotics to select the S17-1 recipient containing recombinant plasmid.S17-1 was directly conjugated with Xanthomonasstrain. S17-1 strain containing recombinant plasmid (3 ml) and recipient Xanthomonasstrain (100 ml) was grown overnight with appropriate antibiotics. Cells were harvested and washed thrice as mentioned earlier. Xanthomonasstrain was finally dissolvedin 600-700 μl sterile water and S17-1 strain was dissolved in 3 ml sterile water. 50 μl Xanthomonascell suspension and 10 μl S17-1 cell suspension were mixed together and 20 μl was spotted on PS agar plate. After 40 h of incubation at 28°C, each conjugation drop was dissolved in 400 μl water separately and plated on PS agar medium with rifampicin (counter-selectable marker) and plasmid specific antibiotics for specific selection of Xanthomonascolony with recombinant plasmid

Since compatible conjugation does not exist between Xanthomonasand E.coliDH5α strain.Therefore, upon getting the appropriate clones in DH5α, conjugation was performed with S17-1 (recipient strain) and PRK600 (helper strain). All the three strains (DH5α with clone, S17-1 and PRK600 strain of E.coli) were grown overnight at 37°C with constant shaking at 200 rpm in 3 ml LB broth. Cells from 1 ml overnight grown cultures were harvested by centrifugation followed by three washes with s